Abstract: Bendamustine is an increasingly used hybrid alkylating agent that is active in lymphoid neoplasias via a novel mechanism of action. There are some pending questions about its use in clinical practice because of its developmental features. A consensus panel of several leading Spanish hematologists with broad experience in the clinical use of bendamustine has established recommendations for the management and treatment of hematological patients with bendamustine based on available clinical data and the experience of the participants. These recommendations address the dose and treatment regimen for different clinical indications, the management of toxicity, and support therapy. This article contains the conclusions of this consensus panel, which are intended to serve as guidelines for the use of bendamustine.

Abstract: Therapeutic approaches against multiple myeloma ( MM ) have largely changed during the past decade. Hematopoietic stem cell transplantation ( HSCT ) and licensing of immunomodulators and proteasome inhibitors have resulted in better response and increased overall survival rates compared to previous conventional therapies. To assess the impact that these new strategies have had on outcome of patients with symptomatic MM in S pain, we conducted an epidemiological retrospective analysis of 338 newly diagnosed patients with stage II ‐ III MM who started first‐line treatment over a 2‐yr period (2003–2005) by collecting data from their medical records. Most patients had been diagnosed with secretory MM (94.4%), 41.7% stage II and 58.3% stage III . The presence of bone lesions (72.2%), as well as anemia (79.8%) and elevated beta2‐microglobulin levels (62.3%), was a common finding; in contrast, hypercalcemia and elevated serum creatinine were less frequent (25% each). First‐line treatment had consisted of either conventional chemotherapy (62%) or induction treatment plus autologous HSCT (38%), as per standard clinical practice. HSCT not only resulted in greater objective response rates (93% vs. 50%), but also contributed to a significant increase in 3‐yr survival (85% vs. 49.7%; 95% CI , range 77–91 vs. 41–58; P 〈 0.001). Overall, 55% of patients presented treatment‐related adverse events, mainly hematological. Toxicity rates were higher among patients treated with alkylating‐based regimens and in those undergoing transplantation. In conclusion, data analysis shows an adequate balance between increased response rates and safety that supports the use of up‐front high‐dose HSCT therapy in younger patients. Most importantly, this study provides further confirmation that the introduction of HSCT has significantly prolonged survival of patients with MM .

Abstract: Next-generation sequencing (NGS) has revealed new insight into the complexity of clonal and subclonal architecture of multiple myeloma (MM). The introduction of targeted studies allows the detection of mutations with very low allele frequencies at affordable price. However, there is few information about the prognostic impact of this mutational profile in series of homogeneously treated MM patients. In this study, we analyzed the most frequently mutated genes in MM from patients included in a randomized clinical trial applying the highest read depth to date. We performed a correlation study between our results and cytogenetics aberrations and clinical outcomes. We used Ampliseq Library Kit 2.0 to target 77 genes (M3Pv2.0 panel) related to critical pathophysiological pathways, associated to drug resistance or targetable with molecular drugs in MM. We sequenced DNA (15ng) from CD138+ purified plasma cells from 80 MM patients at diagnosis included in GEM2010MAS65 clinical trial. We sequenced at an average read depth of 1500X and using Ion Proton sequencer (Thermo Fisher, USA, PaloAlto, CA). We used Ion Reporter software applying in-house modifications in call variants process. We also sequenced DNA from CD138 negative population in 30% of patients in order to filter out potential artifacts and stablish conditions for excluding germline variants. We first fitted a cox-proportional hazard model to predict survival and a second approach using a penalized regression LASSO. We identified 137 gene mutations in the 80 patients analyzed, 54 mutations were predicted pathogenic by Shif and Poliphen and 65 have been described in COSMIC database. 85% of patients harbored at least 1 mutation. The most frequently mutated genes were KRAS (16%), DIS3 (15%), NRAS (13%), BRAF (6%), and TP53 (5%), accounting for the 54% of the total number of mutations. The most frequently mutated pathways were RAS and NFKB in 34% and 28% of patients respectively. Mutational allele frequency for KRAS, BRAF and TP53 was, in all cases, lower than 50% while DIS3 showed mutations in a broad range (from 2 to 85%). Only one patient presented one mutation in NRAS at 73 % of allele frequency. For patients with more than one mutation, two different scenarios have been found. Some patients showed several genes mutated in a similar allele frequency values. On the contrary, a complex subclonal structure was confirmed in 3 patients, with mutations at very different clonal frequency. CD138 negative fraction was sequenced to confirm that these mutations were exclusive of plasma cells. The first of them had two mutations in DIS3 at 8 and 53% of allele frequency, second in KRAS and DIS3 at 13 and 63% respectively and the third in NRAS and IFNGR2 at 5 and 37% respectively. We found no differences in the survival based on pathway, mutational allele frequency or number of mutations. We also investigated pairwise associations between events and we not found significant correlations (Figure 1). Cox-proportional hazard model only showed a negative impact in survival of patients with TP53 mutations. This ultra-deep targeted sequencing strategy is able to detect mutations in most of patients, at very low allele frequency and using a minimal amount of DNA. Despite of its huge capacity to detect mutations, we have not identified new prognostic factors in MM patients treated with highly efficient therapies. However, this study again confirms the complexity of the genomic landscape of MM and the great heterogeneity between patients. The role of theses mutations at relapse as well as in the subsequent treatment effectiveness remains unknown and should be preferably investigated in larger cohorts of homogeneously treated MM patients. Figure 1 Figure 1. Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau. Oriol:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stewart:celgene: Consultancy.

Abstract: Nilotinib is a selective BCR-ABL tyrosine kinase inhibitor (TKI) that has been shown to be more active than imatinib at inducing earlier and deeper molecular responses in the frontline treatment of chronic myeloid leukemia (CML; Larson, et al. Leukemia. 2012). ENEST1st (Evaluating Nilotinib Efficacy and Safety in clinical Trials as First-Line Treatment, NCT01061177) is the largest study ever in TKI-treated patients (pts) with CML. Its primary endpoint was the rate of MR4 (defined as BCR-ABL ≤ 0.01% on the International Scale [BCR-ABLIS] or undetectable BCR-ABL in cDNA with ≥ 10,000 ABL transcripts) at 18 mo. Molecular response (MR) was assessed by standardized quantitative polymerase chain reaction (RQ-PCR) in the 14-laboratory network of the European Treatment and Outcomes Study (EUTOS). Methods ENEST1st is a phase 3b, open-label study of nilotinib 300 mg twice daily (BID) in adults with newly diagnosed BCR-ABL-positive CML in chronic phase. MR was assessed every 3 mo by peripheral blood RQ-PCR at 1 of 14 EUTOS laboratories. Following completion of the 18-mo primary efficacy time point, all pts continued to receive treatment and were followed for an additional 6 mo. Results 1160 pts were screened, with 1086 eligible pts treated in 26 European countries. Overall, median age was 53 y (range, 18-91 y); 59.2% of pts were male, 96.9% had typical b2a2 and/or b3a2 BCR-ABL transcripts (intent-to-treat population for MR analyses), and 90.3% were Philadelphia chromosome–positive by bone marrow metaphase cytogenetics. Per protocol, efficacy and safety analyses were conducted in the initial 819 pts (75.4% of enrolled pts) who were on study for 24 mo or discontinued early. In this group, median age was 53 y (range, 18-91 y), and 58.5% were male; Sokal risk scores were low, intermediate, and high in 33.7%, 39.5%, and 18.7% of pts, respectively (8.1% missing). EUTOS scores were low in 83.8% and high in 9.2% of pts (7.0% missing). A total of 658 pts (80.3%) completed 24 mo of treatment; 161 pts (19.7%) discontinued early. The most common reasons for discontinuation included adverse events (AEs; 10.5%), withdrawal of consent (2.7%), and disease progression (as indicated by the investigator; 1.7%). MR4 rate at 18 mo was 43.0% (95% CI, 39.3-46.8). Rates of major MR (MMR, BCR-ABLIS ≤ 0.1%), MR4, and MR4.5 (BCR-ABLIS ≤ 0.0032% or undetectable cDNA with ≥ 32,000 ABL transcripts) at 18 and 24 mo are shown in the Table. Cumulative incidence of MR4 by 18 mo was 50.1%. All pts were followed up for progression or death for 24 months after start of treatment: 7 pts (0.85%) experienced progression to accelerated phase/blast crisis (AP/BC) while on core treatment or after discontinuation. Overall, 12 pts (1.5%) died; 2 of them died following progression to AP/BC. The safety profile of nilotinib was similar to that observed in other frontline studies. The most common AEs of any relationship were rash (21.4%), pruritus (16.8%), and headache (15.0%); most AEs were grade 1-2. Rates of grade 3-4 hematological AEs were low, with thrombocytopenia and neutropenia reported in 8.2% and 2.9% of pts, respectively. Peripheral arterial occlusive disease (PAOD) occurred in 13 pts (1.6%), ischemic heart disease in 31 (3.8%), and ischemic cerebrovascular conditions in 4 (0.5%). Three cases of pain in extremities are under investigation for potential relationship to PAOD. Conclusions ENEST1st confirms that frontline nilotinib results in high rates of deeper and earlier MR and very low rates of progression to AP/BC. By 18 mo, half of pts achieved a response of MR4. This MR4 rate supports the use of frontline nilotinib as a treatment of choice on which to develop treatment-free remission studies. Finally, the molecular response rates measured in a standardized and validated network of 14 EUTOS laboratories in ENEST1st provide prospective confirmation of the centrally reviewed molecular response rates reported in the ENESTnd trial. Disclosures: Giles: Novartis: Consultancy, Research Funding. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Ariad: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy. Mahon:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Brisol Myers Squibb: Consultancy, Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Rosti:Novartis: Consultancy, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Pfizer: Speakers Bureau. Saglio:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Steegmann:Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ossenkoppele:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Echeveste:Celgene: Consultancy; Novartis: Consultancy. Gattermann:Novartis: Honoraria, Research Funding, Speakers Bureau. Griskevicius:Novartis: Consultancy, Research Funding. le Coutre:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria. Masszi:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Rea:Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Richter:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Stentoft:Novartis: Consultancy, Financial support for relevant congress participation Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Danish Regions: Membership on an entity’s Board of Directors or advisory committees. Tulliez:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Schuld:Novartis: Employment, Equity Ownership. Pellegrino:Novartis: Employment. Walasek:Novartis: Employment. Cross:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria. Hochhaus:Novartis: Research Funding; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Pfizer: Research Funding.

Abstract: We report for the first time the biological features of MRD cells in MM and unravel that clonal selection is already present at the MRD stage. MRD cells show a singular phenotypic signature that may result from persisting clones with different genetic and gene expression profiles.

Abstract: Background There is increasing evidence for the clinical benefit of long-term, continuous therapy for NDMM pts. Proteasome inhibitors (PIs) form a backbone of therapy in MM; however, long-term therapy with some PIs may be limited due to toxicity and the need for regular parenteral administration, increasing the treatment burden. Ixazomib, the first oral PI, has been studied in NDMM pts in 4 phase 1/2 studies in which approximately 1 year of induction therapy with once- or twice-weekly ixazomib plus lenalidomide-dexamethasone (IRd), melphalan-prednisone (IMP), or cyclophosphamide-dexamethasone (ICd) was followed by single-agent ixazomib maintenance. Here we report an integrated analysis of pts from these studies who did not undergo autologous stem cell transplant (ASCT) and received ixazomib maintenance therapy. Methods Pts from 4 studies were included: 1) weekly IRd, 12x28-d cycles IRd (weekly ixazomib 1.68-3.95 mg/m² in phase 1, recommended phase 2 dose [RP2D] 4.0 mg; C16005, NCT01217957; ASCT-eligible/-ineligible pts); 2) twice-weekly IRd, 16x21-d cycles IRd (twice-weekly ixazomib 3.0-3.7 mg in phase 1, RP2D 3.0 mg; C16008, NCT01383928; ASCT-eligible/-ineligible pts); 3) IMP, 9x42-d or 13x28-d cycles IMP (weekly/twice-weekly ixazomib 3.0-5.5/3.0-3.7 mg in phase 1, RP2D of weekly ixazomib 4.0 mg; C16006, NCT01335685; ASCT-ineligible pts); 4) ICd, 13x28-d cycles ICd (weekly ixazomib 4.0 mg; C16020, NCT02046070; ASCT-ineligible pts). In all 4 studies, pts completing induction without progressive disease (PD) and, in the IRd studies, not withdrawn for ASCT, could receive single-agent ixazomib maintenance at the last tolerated dose during induction until PD or unacceptable toxicity. Results 121 pts received ixazomib maintenance (25, 18, 35, and 43 in weekly IRd, twice-weekly IRd, IMP, and ICd studies, respectively). Median age was 72 yrs (range 34-90 yrs; 83% aged ≥65 yrs), 62% had IgG myeloma, 47/34/19% had ISS disease stage I/II/III, 40/49% had Eastern Cooperative Oncology Group performance status 0/1. Combined median progression-free survival (PFS) was 33.8 months from time of study entry and 21.4 months from start of maintenance (Figure). Median PFS from the start of maintenance seemed to be longer for the two IRd studies than for IMP, while median PFS for ICd had not yet been reached. 4-yr OS from time of study entry/3-yr OS from the start of maintenance was 82%. Overall response rate was 93% after induction and 94% after maintenance. Best response rate of CR+VGPR after induction was 57% (22% CR), which increased to 63% (35% CR/sCR) after maintenance; 24 pts (20%) improved their response during maintenance (4 pts with CR improved to sCR; 14 VGPR to CR/sCR; 5 partial response [PR] to VGPR/CR; 1 stable disease to PR). Combined mean dose intensity during maintenance was 89.1%. At time of analysis, median duration of maintenance therapy was 2.2 yrs for weekly IRd, 1.0 yr for twice-weekly IRd, and 0.9 yr for IMP and ICd; maximum duration was 4.8, 3.8, 3.9, and 1.6 yrs respectively. 78% of pts had discontinued maintenance, primarily due to PD (55%), and only 7% due to adverse events (AEs). 15% of pts had ixazomib dose reductions during maintenance. Grade ≥3 AEs (48% vs 74%), drug-related grade ≥3 AEs (24% vs 62%), serious AEs (SAEs; 21% vs 43%), and drug-related SAEs (5% vs 19%) were less common during maintenance than during induction. Common grade ≥3 AEs are shown in the Table. Other AEs of clinical interest were also generally similar or less common during maintenance than during induction, including cardiac arrhythmias (13% vs 21%), pneumonia (12% vs 17%), acute renal failure (7% vs 8%), hypotension (4% vs 7%), myocardial infarction (0 vs & lt;1%), and heart failure (2% vs 2%). Only 6 pts (5%) reported a new primary malignancy. There was only 1 on-study death during maintenance, which was not considered related to ixazomib; due to pulmonary edema in an 81-yr old pt with a history of cardiovascular co-morbidities. Conclusions In this integrated analysis, single-agent ixazomib maintenance therapy following an ixazomib-based induction regimen was associated with deepening of responses and good long-term outcomes in NDMM pts not undergoing ASCT. Single-agent ixazomib is feasible for long-term administration, with limited new-onset grade ≥3 AEs. These outcomes appear similar to other studies involving maintenance approaches in NDMM and support the ongoing phase 3 investigation of ixazomib maintenance therapy. Disclosures Dimopoulos: Novartis: Consultancy, Honoraria; Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Genesis Pharma: Research Funding. Laubach: Novartis, Takeda, Celgene, Onyx: Research Funding; Novartis, Takeda, Celgene: Consultancy. Hofmeister: Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Roche: Research Funding; Karyopharm: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Janssen: Research Funding; Celgene: Research Funding. San Miguel: Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kumar: Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Skyline: Honoraria. Lu: Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Teng: Array Pharmaceuticals: Equity Ownership; AMAG Pharmaceuticals: Equity Ownership; Gilead Science: Equity Ownership; Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Liu: Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Byrne: Oncopeptides AB: Consultancy; Takeda: Consultancy. Berg: Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Labotka: Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. van de Velde: Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Richardson: Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees.

Abstract: VMP and Rd are two of the most efficient and widely accepted regimens in the treatment of elderly newly diagnosed MM patients. In order to further improve the outcome of elderly patients, one possibility would be to use regimens including all these drugs simultaneously, but this may result into high toxicity. Alternatively, the use of these regimens (VMP and Rd) in a sequential or alternating scheme could improve the treatment of elderly patients. We hypothesized the alternating scheme would minimize the emergence of resistant clones, and would reduce the cumulative toxicity. In order to test this hypothesis we decided to compare VMP and RD in a sequential vs an alternating scheme. Patients and methods 241 patients were randomized to receive a sequential scheme consisting on 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd (half of the patients started by VMP and half by Rd) up to 18 cycles). VMP included the iv administration of bortezomib 1.3 mg/m2 twice weekly for 1 six-weeks cycle, followed by once weekly for 8 four-weeks cycles in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2 once daily on days 1–4 of each cycle. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. Results 121 patients were allocated to receive the sequential scheme and 120 the alternating regimen. Both arms were well balanced according to the baseline characteristics. 52% patients in the sequential arm and 55% in the alternating and had high risk cytogenetic abnormalities (t(4;14), t(14;16), del17p or 1q gains). After 9 cycles of treatment, in the sequential arm, 35 out of 66 (54%) achieved at least VGPR vs 51 out of 65 patients (78%) in the alternating arm (p=0.002), including sCR/CR rate of 28% vs 38% in the sequential and alternating arms, respectively (p=NS). Seven patients in each arm achieved immunophenotypic CR. Moreover, while four patients progressed in the sequential arm under treatment with VMP, no patients in the alternating scheme developed disease progression during the first 9 cycles, After a median follow up of 12 months, there was a trend for shorter TTP in the sequential as compared with the alternating scheme (18 m-TTP of 83% vs 89% (p=NS)). In terms of OS, 83% of patients in the sequential arm were alive at 18 m versus 93% in the alternating (p=NS). Patients who achieved sCR/CR had a significantly longer 18 m-TTP as compared with patients who didn’t achieve it in both sequential (100% vs 71%; p=0.006) and alternating arms (100% vs 79%; p=0.006) and this translated into a significant benefit in OS. No differences were observed in overall response rates and CR rates in standard and high risk patients. The 18 m-TTP was similar in standard and high risk groups in both sequential (86% vs 81%) and alternating arms (84% vs 94%), noting that 94% of patients receiving the alternating scheme were progression-free at 18 months. Regarding hematologic toxicity, the frequency of G3-4 neutropenia was slightly lower in the sequential than in the alternating arm (16% and 23%) and the same trend was observed for G3-4 thrombocytopenia (16% vs 20%). Concerning non-hematologic toxicity, 5% and 4% of the patients in the sequential and alternating arms developed G3-4 infections, respectively; the rate of G3-4 skin rash was 4% in the sequential and 3% in the alternating arm; 4% of patients in the sequential arm developed G3-4 peripheral neuropathy and 3% in the sequential arm. The rate of grade 3-4 thrombotic events was 2% in both arms. Nevertheless, the detailed evaluation of the toxicity will be done at the completion of the trial when all patients will have received the same amount of drugs in either a sequential or an alternating scheme (at the present time, 42 patients in the sequential arm were not yet at risk for the development of lenalidomide-related side effects). Conclusions The administration of melphalan, bortezomib, lenalidomide and steroids in elderly MM patients in a sequential or alternating scheme is feasible. Although longer follow-up is necessary, the alternating scheme may be superior in terms of response rate and outcome, as result of the early exposure of the plasma cell to different agents. Toxicity profile is acceptable. Aparently both schemes of therapy seems to overcome the poor prognosis of high risk cytogenetic. Disclosures: Mateos: Janssen, Celgene: Honoraria. Off Label Use: Lenalidomide plus dexamethasone is not approved for newly diagnosed MM patients. Ocio:Onyx: Consultancy, Research Funding; Novartis: Consultancy; Array Biopharma: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding. San Miguel:Janssen, Celgene: Membership on an entity’s Board of Directors or advisory committees.

Abstract: The identification of subclones in multiple myeloma (MM) could have clinical implications since the complete eradication of all clones is required to prolong patients’ survival. Hence, there is an unmet need to fully characterize such subclones. In the present study, we started by investigating using multidimensional (23-color) flow cytometry (MFC) the presence of ≥2 distinct bone marrow PC subclones in 116 newly-diagnosed (MM) patients included in the Spanish GEM2010 trial: sequential VMP (9 cycles) followed by Rd (9 cycles) vs alternating VMP with Rd (18 cycles). By principal component analysis (PCA) using the Infinicyt software, the immunophenotypic expression profile (based on the simultaneous evaluation of 23 antigens in single-clonal-PCs) showed the presence of two or more subclones in 35/116 (30%) newly-diagnosed MM patients. The most powerful antigens for the identification of subclonal heterogeneity were CD56, CD27, CD81, CD20, CD33, β7, CD138, CD49e, CD54, HLADR, CD19, CD44 and CXCR4. Then, we investigated the genetic profile of distinct phenotypic subclones (sorted by FACS with purity ≥97%) through high-density Cytoscan750K (n=5) arrays and FISH (n=8). In 2/5 (40%) patients, specific copy number abnormalities (CNA; only those with minimum of 25 consecutive imbalanced markers per segment and minimum 100 Kb length were considered) were present in one clone [del(13q), del(14q), del(20p), +(20q), +(21q), or del(22q)] but absent in the other. Similar results were observed by FISH while investigating those probes used as part of the clinical workout. Noteworthy, in three cases del(17p13) was detected in only one subclone, whereas del(13q14) was restricted to one subclone in two patients. To assess the clonogenic potential of distinct phenotypic subclones detected in the whole malignant PC compartment, these were sorted by MFC and seeded in coculture with the human mesenchymal cell line hTERT (with Methocult® supplemented with lymphocyte conditioned medium (10%) plus 20 ng/ml IL-6 plus 20 ng/ml IGF-1). In 2 out of 7 patients (29%) we observed different clonogenic potential between two patient-specific subclones (patient 1, 208 vs 0 colonies; patient 2, 5 vs 1 colony). Finally, we aimed to investigate if distinct phenotypic subclones display different chemoresistance. To address this question, we compared the immunophenotypic expression profile of paired baseline vs MRD clonal PC in 9 patients enrolled in the Spanish GEM2010 trial in which ≥2 subclones were identified at diagnosis. Our results show a phenomenon of clonal restriction in 7/9 (78%) patients after chemotherapy, indicating that among the initial tumor bulk, only specific subclones are primary chemoresistant (i.e.: MRD) in patients otherwise achieving a serological response. As an example, in one patient showing at diagnosis four clonal PC subsets (CD33-/CD117-, CD33+/CD117-, CD33+/CD117+ and CD33+/CD117+), only one subclone (CD33-/CD117-) persisted after 9 cycles of alterning Rd-VMP. In conclusion, our results show that distinct phenotypic subclones translate into a different cytogenetic profile and clonogenic potential. Since the balance between different subclones may change over the course of treatment, so may change the extent of specific cytogenetic abnormalities and the patient risk. Thus, careful monitoring of specific subclones may have implications towards an adapted therapy to overcome chemoresistant cells. This work was supported by funding from the RTICC-Hematology Group (RD12/0036/0058), the Asociación Española Contra el Cáncer (AECC) (GCB120981SAN) and Multiple Myeloma Research Foundation (MMRF) 2012 Research Fellow Award (B Paiva) Disclosures: No relevant conflicts of interest to declare.